Vitamin Analysis in High Performance Liquid Chromatography (HPLC)

Procedure: Oil extracted from fish meat as described (Folch et al., 1957) earlier under fatty acids was used for analysis of fat soluble vitamins. About 0.15 g fish oil was refluxed with 25 mL methanol and 150% potassium hydroxide (KOH) in water bath for 30 min. Fat soluble vitamins was extracted with 50 mL petroleum ether. The petroleum ether layer was collected, concentrated and dissolved in 5mL acetonitrile (ACN). Fat soluble vitamins were analyzed by injecting 20 µL of sample in HPLC (Shimadzu LC 10AS) equipped with C18 RP column and UV detector.10 The mobile phase of HPLC consisted of ACN (solvent A) and methanol (solvent B). A simple linear gradient system was used, starting from (solvent A/solvent B) 50/50 to 70/30 in 20 min. The mobile phase flow rate was 1 mL/min. The fat soluble vitamins A, D, E, K were identified and quantified by comparing retention times and peak area with those of vitamins standards respectively (R7632, C9756, T3251 and M5625, Sigma-Aldrich).

Reference

• 1. Folch J, Lees M and Sloane-Stanley GH (1957) A simple method for the isolation and purification of total lipids from animal tissues. Journal of Biological Chemistry 226: 497-509

• 2. Metcalfe LD, Schmitz AA and Petha JR (1966) Rapid preparation of fatty acid esters from lipids for gas chromatography analysis.Analytical Chemistry 38:514-517.

• 3. Mohanty BP, Paria P, Das D, Ganguly S, Mitra P, Verma A, et al, (2012) Nutrient profile of giant river-catfish Sperata seenghala (Sykes). National Academy of Science Letter 35:155-161.